Project Summary/Abstract This career development project is focused on orthopedic training and conducting novel research into the role of thiol metabolism during chronic oxidative stress after mechanical injury to establish an independent line of research. This will be done under the guidance of: primary mentor, Dr. James Martin, an expert in orthopedic cell biology with over 30 years' experience in research and training; co-mentor for redox biology, Dr. Douglas Spitz, an expert in redox biology with 30 years of continuous independent NIH funding and training experience including an NIH T32 and several K-award mentorships; and clinical co-mentor, Dr. Larry Marsh, a world class trauma surgeon and clinical researcher with experience training clinical and basic scientists. These mentors will guide the acquisition of preliminary data to characterize chondrocyte thiol metabolism and redox signaling, the search and negotiation for a tenure track position, the execution of the R00 research into thiol metabolism after injury, and final R01 composition to establish independence. The training goal of the K99 phase of this project is to provide critical knowledge and experience for operation as an independent orthopedic researcher via incorporation into resident training. Resident conferences will be attended including no fewer than three days a week, 1-2 hours each day. The distinguished University of Iowa Department of Orthopedics and Rehabilitation provides a powerful research environment for pursuit of research goals during the K99 phase, highlighted by large animal models of orthopedic disease. Concurrent with their funded projects and using the same animals, the redox status of intracellular thiols including glutathione and mitochondrial thioredoxin/peroxiredoxin, NRF2/Bach-1 interplay, GPx4 content, and mitochondrial redox function during disease development after intraarticular fracture will be measured. These endpoints have not been explored as a unified pathway, and have not been described in these disease settings. Observing these pathways over three different time points corresponding to early, intermediate, and late stages of disease in a large animal model will allow for a comprehensive understanding of the status of intracellular thiols over time. Taken together with the clinical training described, this will result in an enhanced orthopedic skill set, and development of novel research ideas in well characterized orthopedic models. Detailed description of thiol redox responses during disease will enable logical design of an adeno-associated virus seeking to disrupt thiol metabolism similar to chronic orthopedic disease processes. As the R00 phase commences, data from the K99 phase will guide construction and characterization of the viral tool described. Once this tool has been validated, it will be applied in a rabbit model of post-traumatic injury and complimented with a transgenic mouse resistant to oxidative stress. By comparing responses to injury from control rabbits to rabbits overexpressing Bach-1, a mechanistic role for thiol metabolism in response to inflammation will be described.